Discovery of human antibodies to cell surface antigens by capture lift screening of phage-expressed antibody libraries.
An assay for the rapid identification and cloning of antibody fragments (Fabs) reactive with cell surface antigens was established and used to identify Fabs selectively reactive with tumor cell surface antigens. The Fabs were produced by a phage expression system and screened by a modified plaque lift approach in which nitrocellulose filters were coated with an anti-immunoglobulin reagent and blocked with bovine serum albumin prior to application to the phage-infected bacterial lawn. Subsequently, capture lifts were incubated with biotinylated antigen and reactive Fabs were identified with streptavidin conjugates. This screening method, termed capture lift, results in the immobilization of greater quantities of Fab and decreases the binding of unrelated host proteins, resulting in a more sensitive plaque lift assay. The capture lift permits the simultaneous analysis of thousands of antibody clones and, more importantly, can be used with crude detergent-solubilized cell extracts, permitting the discovery of Fabs which bind integral membrane proteins present in heterogeneous mixtures of antigens. Optimal conditions were identified utilizing phage-expressed BR96 Fab and a horseradish peroxidase conjugate of Lewis Y, a soluble cross-reactive antigen. Subsequently, it was demonstrated that the assay was functional with postnuclear detergent extracts isolated from surface-biotinylated tumor cells expressing the BR96 tumor antigen. Purification of the target antigen was not required. To demonstrate the application of the capture lift assay for the discovery of Fabs reactive with novel cell surface antigens a phage-expressed human antibody library constructed from tumor-infiltrating B lymphocytes was screened. Multiple antibody clones which reacted with detergent-solubilized biotinylated surface antigens were identified. Upon further characterization a portion of these displayed selectivity for tumor cells, as demonstrated by the binding of Fab to fixed and live tumor cells but not normal fibroblasts.
Watkins
J D
JD
Ixsys, Inc., San Diego, California 92121, USA.
Beuerlein
G
G
Wu
H
H
McFadden
P R
PR
Pancook
J D
JD
Huse
W D
WD
D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology*; D017104:Bacteriophage M13 / Q000276:immunology; D019921:Biotinylation; D002460:Cell Line; D002846:Chromatography, Affinity; D003101:Collodion; D004797:Enzyme-Linked Immunosorbent Assay; D005374:Filtration / Q000295:instrumentation; D005434:Flow Cytometry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000302:isolation & purification*; D051379:Mice; D019151:Peptide Library; D012815:Signal Processing, Computer-Assisted; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D003101:Collodion